2.3 Cell culture
Human umbilical vein endothelial cells (HUVECs) were purchased from Lonza and cultured in endothelial cell growth medium-2 (EGM-2, Lonza, Cat#CC-3156 & CC-4176) at 37 °C in 5% CO2 before the experiment. Subconfluent cells obtained after 5-7 passages were used in the following experiments. Twelve h prior to cell culture, culture media was removed and replaced with phenol red-free low-glucose DMEM (Gibco, Cat#11054020) supplemented with 1% calf serum (Gibco, Cat#16010159). HUVECs were then transferred to EGM-2 with high glucose (HG, 33 mM) in the presence of FGF2 or PEG-FGF2 conjugates (10 ng·mL-1) for 72 h. PBS (Gibco, Cat#10010) was used as a negative control (untreated cells). The media was replaced every 24 h. All experiments were independently carried out at least three separate times.
Human dermal fibroblasts (HDFs) were separated from human foreskin samples obtained from volunteers, as previously described (Takahashi et al. , 2006). HDFs were cultured in DMEM supplemented with 5.5 mM glucose, 10% FBS, and 1% penicillin-streptomycin, and were passaged by using 0.25% trypsin (Gibco) when cell confluence reached ~80%. Primary human fibroblasts at passage 5-6 were used in the experiments described below.